Disclosed are benzothiazin derivatives of formula (I), preparation methods therefore, and treatment methods employing such compounds as antibacterial agents in treating infectious diseases of mammals caused by bacteria, especially diseases like tuberculosis and leprosy caused by mycobacteria: [structure] wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 , and other variables enumerated under one or more of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are as defined.

Description

17 parts

›BACKGROUND OF THE INVENTION

The present invention relates to novel benzothiazin derivatives and their use as antibacterial agents in infectious diseases of mammals (humans and animals) caused by bacteria, especially diseases like tuberculosis (TB) and leprosy caused by mycobacteria.

Thiazinone, their derivatives and their use as antibacterial agents, especially against mycobacteria (TB), laid open for public in AR 24 25 67 A1, AU 37 04 400 A1, CA 13 22 551 C1 or EP 0 245 901 B1 for instance.

As known, there is a threadful worldwide increase in tuberculosis infections with mycobacteria which developed resistance against the available therapeutics (B. R. Bloom, J. L. Murray, tuberculosis: commentary on a reemergent killer. Science 257, 1992, 1055-1064). Extremely dangerous is the development of multidrug resistant (MDR) mycobacteria. These are mycobacteria, resistant at least against two of the most active tuberculosis drugs, isoniazid and rifampicin, but also against streptomycin, pyranzinamid and ethambutol. The proportion of MDR-TB in some countries is already more than 20%. Together with the increased number of TB diseases generally, worldwide it causes about 3,000,000 deaths annually.

For the treatment of such diseases, like (TB) or leprosy there is an urgent need for new drugs with new mechanisms of actions, especially to overcome drug resistance and to overcome the known dramatic side effects of the available drugs.

›SUMMARY OF THE INVENTION · 1 of 2

The present invention aims at the generation of new compounds with activity against mycobacteria as potential new tuberculosis drugs to overcome problems concerning resistance and drug intolerance.

This aim has been solved by providing compounds of the formula I

Wherein R 1 and R 2 are, independently each from other, NO 2 , CN, CONR 7 R 8 , COOR 9 , CHO, halogen, NR 7 R 8 , SO 2 NR 7 R 8 , SR 9 , OCF 3 , mono-, di or trifluoromethyl;

R 3 and R 4 are, independently each from other, H, a saturated or unsaturated, linear or branched aliphatic radical having 1-7 chain members, cycloalkyl having 3-6 carbon atoms, benzyl, SR 9 , OR 9 ;

R 5 and R 6 are, independently each from other, a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-8 chain members, cycloalkyl having 3-6 carbon atoms, phenyl, or R 5 and R 6 together represent a bivalent radical —(CR 9 2 ) m —, or R 5 and R 6 together represent bivalent radicals:

wherein m is 1-4, or represent bivalent radicals a saturated or unsaturated mono or polyheterocycles with heteroatoms N, S, O and substituted by (R 10 )x, wherein x is 1-4;

R 7 , R 8 and R 9 are, independently each from other H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members, mono-, di or trifluoromethyl, halogen, phenyl, or R 3 and R 4 together represent a bivalent radical —(CH 2 ) n — wherein n is 2-7;

R 10 is H or a saturated or unsaturated, halogenated or unhalogenated, linear or branched aliphatic radical having 1-7 chain members, NO 2 , NR 7 R 8 , CN, CONR 7 R 8 , COOR 9 , CHO, halogen, SO 2 NR 7 R 8 , SR 9 , OR 9 , OCF 3 , mono-, di or trifluoromethyl, benzyl or phenyl.

In a preferred embodiment the invention concerns compounds of the formula (I) selected from the group consisting of

2-(4-R 5 -4-R 6 -piperidin-1-yl)-8-nitro-6-trifluoromethyl-1,3-benzothiazin-4-one, 6-cyano-2-(4-R 5 -4-R 6 -piperidin-1-yl)-8-nitro-1,3-benzothiazin-4-one, 6-amido-2-(4-R 5 -4-R 6 -piperidin-1-yl)-8-nitro-1,3-benzothiazin-4-one, 2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-R 1 -6-R 2 -1,3-benzothiazin-4-one, 2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-R 1 -6-R 2 -1,3-benzothiazin-4-one, 2-[(2R)-2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-R 1 -6-R 2 -1,3-benzothiazin-4-one, 2-[(2S)-2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-R 1 -6-R 2 -1,3-benzothiazin-4-one, 2-(2,3-dimethyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-R 1 -6-R 2 -1,3-benzothiazin-4-one, 2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-R 1 -6-R 2 -1,3-benzothiazin-4-one,

wherein R 1 , R 2 , R 5 and R 6 have the above meanings,

The present invention is even more particularly concerned with at least one compound selected from the group consisting of

2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one, 2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one, 2-(4,4-diethoxypiperidin-1-yl)-6,8-dinitro-1,3-benzothiazin-4-one, 7-methyl-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one, 2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one, 2-(2,3-dimethyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one, 2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one, 2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile, 2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile, 8-amino-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-4-oxo-1,3-benzothiazine-6-carbonitrile and 8-amino-2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6-(trifluoromethyl)-1,3-benzo-thiazin-4-one.

For the synthesis of the aimed compounds we developed our original method of 1,3-benzothiazin-4-one synthesis with usage of dithiocarbamate derivatives as intermediate (method A). The classical method of 1,3-benzothiazin-4-one synthesis with usage of thiocyanate salts (method B) is usable too. Both are presented in the scheme below.

Surprisingly the compounds of the invention exhibit strong antibacterial activity, especially against mycobacteria with minimal inhibitory concentrations (MIC) in the range of 0.23 pg/ml->10 μg/ml for fast growing mycobacteria, of 0.195-1.56 μg/ml for M. tuberculosis , including multiresistant strains determined by the classical method and of 0.030 μg/ml for M. tuberculosis H37Rv determined by the Alamar Blue method. Surprisingly the compounds of the invention demonstrate a high level of selectivity for mycobacteria only which reduces the potential for adverse side effects dramatically.

The compounds of the invention are non-mutagenic at 5 mg/ml in the SOS chromotest.

The compounds of the invention are in vivo therapeutically active in the murine model of tuberculosis infection superior compared to the main antituberculosis drug isoniazid used as a positive control. 100% of mice survived. All control animals died until day 33.

The compound of the invention (especially compound no 2=example 1 in the embodiments), is non toxic after per os administration of doses ranging up to 2000 mg/kg was the compound was well endured by animals in the first and 24 next coming hours after introducing. During 7 days of investigations the compound 2 did not cause changes in general state and behavior of the mice, it did not affect motor and reflex activity, active and calm cycles, grooming, food consumption, there were no cases of animal death. LD 50 for compound 2 is >2000 mg/kg.

Thus, the compounds of the invention are useful for the treatment of tubercular infection and other mycobacterial infections, in humans and in animals.

Accordingly, the invention concerns pharmaceutical compositions comprising a compound of the formula I.

The invention relates furthermore to a compound of the formula I for use in a method for the treatment of bacterial infections in mammals. Preferred compounds of the formula I for use in such method are those specifically listed above.

›SUMMARY OF THE INVENTION · 2 of 2

The compounds of the invention are formulated for use by preparing a dilute solution or suspension in pharmaceutically acceptable aqueous, organic or aqueous-organic medium for topical or parenteral administration by intravenous, subcutaneous or intramuscular injection, or for intranasal application; or are prepared in tablet, capsule or aqueous suspension form with conventional excipients for oral administration or as suppositorium.

The compounds can be used in dosages from 0.001-1000 mg/kg body weight.

The examples which follow in the subsequent experimental part serve to illustrate the invention but should not be construed as a limitation thereof.

The structures of the compounds of the invention were established by modes of synthesis and elementary analysis, and by nuclear magnetic resonance and/or mass spectra, as well as by X-ray analysis.

›DETAILED DESCRIPTION OF THE INVENTION

Starting Materials

Chemicals and solvents were purchased from Lancaster Synthesis (Lancashire, England) or from Aldrich (Sigma-Aldrich Company, St-Louis, US) and were used in the synthesis without additional purification. Melting points were determined according to the BP procedure and are uncorrected (Electrothermal 9001, GB). If analyses are indicated only by the symbols of the elements, analytical results are within ±0.3% of the theoretical values (Carlo-Erba 5500, Italy). NMR spectra were determined with a Varian Unity Plus 400 (USA). Shifts for 1 H NMR are reported in ppm downfield from TMS (δ). Mass spectra were obtained using a Finnigan SSQ-700 (USA) instrument with direct inject. Reactions and purity of compounds were controlled by TLC with usage Silicagel 60 F 254 aluminium sheets (Merck Co, Germany).

›Examples13

›Example 1

2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one, (compound 1)

Method A.

To a stirred 50 mL solution of 25% aqueous ammonia was added drop-wise a solution of 5 g of 2-chloro-3-nitro-5-trifluoromethylbenzoyl chloride (D. E Welch, R. R. Baron, B. A. Burton, J. Med. Chem. 12; 2; 1969; 299-303) in acetonitrile (10 mL) at −20° C. 10 min later, 50 ml of ethyl acetate was added. The organic phase was separated, washed twice in water, dried over Na 2 SO 4 , treated by activated carbon, filtered and concentrated in vacum. The crude product was purified by crystallization from ethanol. The yield of 2-chloro-3-nitro-5-(trifluoromethyl)benzamide was 92%. mp 195-197° C. (methanol).

Anal. Calcd. for C 8 H 4 ClF 3 N 2 O 3 : C, 35.78; H, 1.50; N, 10.43

Found: C, 36.01; H, 1.53; N, 10.39

0.5 g of 2,2-chloro-3-nitro-5-(trifluoromethyl)benzamide was dissolved in a 25 ml of ethanol. The reaction mixture was treated with of 0.5 g of 1,4-dioxa-8-azaspiro[4.5]decane-8-carbodithioic acid sodium salt dihydrate (Z. Ge, R. Li, T. Cheng, Synth. Commun., 29, 18, 1999, 3191-3196) and stored for 18 h at room temperature. It was then poured into 50 ml of cooled water and the resulting yellow precipitate was filtered off. Pure final product was obtained after recrystallization twice from ethanol. 2-(Aminocarbonyl)-6-nitro-4-(trifluoromethyl)phenyl-1,4-dioxa-8-azaspiro[4.5]decane-8-carbodithioate is light yellow crystalline solid. Yield 0.47 g %. mp 138-140° C.

Anal. Calcd. for C 11 H 12 N 4 O 2 S 2 : C, 42.57; H, 3.57; N, 9.31; S, 14.21

Found: C, 42.61; H, 3.67; N, 9.22; S, 14.30

0.4 g of 2-(aminocarbonyl)-6-nitro-4-(trifluoromethyl)phenyl-1,4-dioxa-8-azaspiro[4.5]decane-8-carbodithioate was dissolved in a 25 ml of ethanol. The reaction mixture was treated with of 0.32 g of Na 2 HPO 4 ×12H 2 O and refluxed for 6 h. It was then cooled and light yellow precipitate was filtered off and washed by 30 ml methanol. Pure final product was obtained after recrystallization twice from ethanol. 2-(1,4-Dioxa-8-aza-spiro[4.5]dec-8-yl)-8-nitro-6-trifluoromethyl)-1,3-benzothiazin-4-one is light yellow crystalline solid. Yield 0.47 g %. mp 211-212° C.

R f ((hexane-acetone; 2/1)-0.35

MS m/z 417 (M + ).

1 H NMR (DMSO-d 6 ) δ 8.83 and 8.77 (two 1H, two s, 2CH), 3.80 (8H, broad s, N(CH 2 CH 2 ) 2 C), 2.02 (4H, broad s, OCH 2 CH 2 O) ppm.

Anal. Calcd. for C 16 H 14 F 3 N 3 O 5 S: C, 46.04; H, 3.38; N, 10.07; S, 7.68

Found: C, 45.94; H, 3.37; N, 10.09; S, 7.76

Method B. The procedure in detail was the same as described in J. Imrich, P. Kristian, Coll. Czech. Chem. Commun., 47, 1982, 3268-3282; D. Koscik, P. Kristian, J. Gonda, E. Dandarova, Coll. Czech. Chem. Commun., 48, 1983, 3315-3328; D. Koscik, P. Kristian, O. Forgac, Coll. Czech. Chem. Commun., 48, 1983, 3427-3432; T. H. Cronin, H.-J. E. Hess, U.S. Pat. No. 3,522,247. Yield of 2-(1,4-dioxa-8-aza-spiro[4.5]dec-8-yl)-8-nitro-6-trifluoromethyl)-1,3-benzo-thiazin-4-one is 0.21%. The compound is identical by spectroscopical data to the compound synthesized by method A.

›Example 2

2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoro-methyl)-1,3-benzothiazin-4-one, (compound 2)

Following the procedure of Example 1. Light yellow crystalline solid.

Yield 54%. mp 192-3° C.

R f (hexane-acetone; 2/1)-0.30.

MS m/z 431 (M + ).

1 H NMR (DMSO-d 6 ) δ 8.81 and 8.77 (two 1H, two s, 2CH), 4.24 (1H, m, CH), 4.11 (1H, m, CH), 4.06 (4H, broad s, N(CH 2 ) 2 ), 3.47 (1H, t, CH), 3.27 (1H, s, CH), 1.80 (4H, broad d, C(CH 2 ) 2 ), 1.23 (3H, d, CH 3 ) ppm.

Anal. Calcd. for C 17 H 16 N 3 O 5 S: C, 47.33; H, 3.74; N, 9.74; S, 7.43

Found: C, 47.36; H, 3.80; N, 9.87; S, 7.51

›Example 3

2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-6,8-dinitro-1,3-benzothiazin-4-one, (compound 4)

Following the procedure of Example 1 with usage of 2-hydroxy-3,5-dinitrobenzoic acid as starting material. Light yellow crystalline solid.

Yield 43%. mp 271-3° C. (EtOH/DMF).

R f (hexane-acetone; 2/1)-0.25.

MS m/z 394 (M + ).

1 H NMR (DMSO-d 6 ) δ 9.15 and 9.12 (two 1H, two s, 2CH), 3.86 (8H, broad s, N(CH 2 CH 2 ) 2 C), 2.97 (4H, broad s, OCH 2 CH 2 O) ppm.

Anal. Calcd. for C 15 H 14 N 4 O 7 S: C, 45.68; H, 3.58; N, 14.21; S, 8.13

Found: C, 45.34; H, 3.56; N, 14.30; S, 7.98

›Example 4

2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]decyl)-6,8-dinitro-1,3-benzothiazin-4-one, (compound 4)

Following the procedure of Example 1 with usage of 2-hydroxy-3,5-dinitrobenzoic acid as starting material. Yellow crystalline solid. Yield 57%. mp 139-142° C. (EtOH/DMF).

R f (hexane-acetone; 2/1)-0.50.

MS m/z 408 (M + ).

1 H NMR (DMSO-d 6 ) δ 9.08 and 9.11 (two 1H, two s, 2CH), 4.23 (1H, m, CH), 4.10 (1H, m, CH), 4.06 (4H, broad s, N(CH 2 ) 2 ), 3.43 (1H, t, CH), 3.27 (1H, s, CH), 1.80 (4H, broad d, C(CH 2 ) 2 ), 1.20 (3H, d, CH 3 ) ppm.

Anal. Calcd. for C 16 H 16 N 4 O 7 S: C, 47.06; H, 3.95; N, 13.72; S, 7.85

Found: C, 46.87; H, 3.91; N, 13.57; S, 7.83

›Example 5

2-(2,3-dimethyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one, (compound 5)

Following the procedure of Example 1 with usage of 2-hydroxy-3-nitro-5-trifluoromethylbenzoic acid as starting material. Light yellow crystalline solid. Yield 58%. mp 205-207° C. (EtOH/DMF).

R f (hexane-acetone; 2/1)-0.55.

MS m/z 44522 (M + ).

1 H NMR (DMSO-d 6 ) δ 8.82 and 8.77 (two 1H, two s, 2CH), 3.86 (4H, broad c, N(CH 2 ) 2 ), 3.45-3.53 (2H, m, 2CH), 2.41 (4H, broad d, C(CH 2 ) 2 ), 1.13-1.17 (6H, m, 2CH 3 ) ppm.

Anal. Calcd. for C 18 H 18 F 3 N 3 O 5 S: C, 48.54; H, 4.07; N, 9.43; S, 7.20

Found: C, 48.66; H, 4.12; N, 9.32; S, 7.46

›Example 6

2-(4,4-diethoxypiperidin-1-yl)-6,8-dinitro-1,3-benzothiazin-4-one, (compound 6)

Following the procedure of Example 1 with usage as starting material 2-hydroxy-3,5-dinitrobenzoic acid. Yellow crystalline solid. Yield 32%.

mp 179-181° C. (i-PrOH).

R f (hexane-acetone; 2/1)-0.30.

MS m/z 424 (M + ).

1 H NMR (DMSO-d 6 ) δ 9.08 and 9.11 (two 1H, two s, 2CH), 3.60-3.67 (4H, m, N(CH 2 ) 2 ) 2.11-2.08 (4H, m, C(CH 2 ) 2 ), 3.47 and 3.57 (two 2H, q, 2OCH 2 ), 1.16 (6H, t, 2CH 3 ), ppm.

Anal. Calcd. for C 17 H 20 N 4 O 7 S: C, 48.11; H, 4.75; N, 13.20; S, 7.56

Found: C, 48.12; H, 4.73; N, 13.41; S, 7.67

›Example 7

2-(7,12-dioxa-3-azaspiro[5.6]dodec-3-yl)-6,8-dinitro-1,3-benzothiazin-4-one, (compound 7)

Following the procedure of Example 1 with usage as starting material 2-hydroxy-3,5-dinitrobenzoic acid. Yellow crystalline solid. Yield 51%.

mp 193-195° C. (i-PrOH/DMF).

R f (hexane-acetone; 2/1)-0.45.

MS m/z 422 (M + ).

1 H NMR (DMSO-d 6 ) δ 8.97 and 9.16 (two 1H, two s, 2CH), 3.57-3.74 (8H, m, 4CH 2 ), 1.93-2.35 (8H, m, 4CH 2 ) ppm.

Anal. Calcd. for C 17 H 18 N 4 O 7 S: C, 48.34; H, 4.30; N, 13.26; S, 7.56

Found: C, 48.21; H, 4.43; N, 13.30; S, 7.66

›Example 8

2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-7-methyl-6,8-dinitro-1,3-benzothiazin-4-one, (compound 8)

Following the procedure of Example 1 with usage as starting material 2-hydroxy-4-methyl-3,5-dinitrobenzoic acid. Yellow crystalline solid.

Yield 51%. mp 207-210° C. (i-PrOH/DMF).

R f (hexane-acetone; 2/1)-0.30.

MS m/z 408 (M + ).

1 H NMR (DMSO-d 6 ) δ 8.77 (1H, s, CH), 3.86 (8H, broad s, N(CH 2 CH 2 ) 2 C), 2.97 (4H, broad c, OCH 2 CH 2 O), 2.79 (3H, s, CH 3 ) ppm.

ppm.

Anal. Calcd. for C 16 H 16 N 4 O 7 S: C, 47.06; H, 3.95; N, 13.72; S, 7.85

Found: C, 47.12; H, 4.01; N, 13.69; S, 7.94

›Example 9

2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile, (compound 9)

To a stirred solution of 5 g (19 mmol) 2-hydroxy-5-iodobenzoic acid in 50 ml DMF was added by small portions dry 2.5 g (22 mmol) of CuCN (I). The reaction mixture was refluxed during 5 h, 100 ml of water and 50 ml ethylacetate were added. After it conc. Hydrochloric acid was added up to pH ˜3 very carefully under good ventilation. The organic phase was separated, washed twice in water, dried over Na 2 SO 4 , treated by activated carbon, filtered and concentrated in vacum. The crude product was purified by crystallization from water. The yield of 5-cyano-2-hydroxybenzoic acid was 71%. Following the procedure of Example 1. Yield 44%. mp 217-220° C. (EtOH/DMF).

R f (hexane-acetone; 2/1)-0.50.

MS m/z 374 (M + ).

1 H NMR (DMSO-d 6 ) δ 8.74 and 8.67 (two 1H, two s, 2CH), 3.41 (8H, broad s, N(CH 2 CH 2 ) 2 C), 2.93 (4H, broad s, OCH 2 CH 2 O) ppm.

Anal. Calcd. for C 16 H 14 N 4 O 5 S: C, 51.33; H, 3.77; N, 14.97; S, 8.57

Found: C, 51.30; H, 3.84; N, 14.89; S, 8.62

›Example 10

2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile, (compound 10)

Following the procedure of Example 9. Yellow crystalline solid. Yield 34%. mp 251-253° C. (EtOH/DMF).

R f (hexane-acetone; 2/1)-0.40.

MS m/z 388 (M + ).

1 H NMR (DMSO-d 6 ) δ 8.73 and 8.61 (two 1H, two s, 2CH), 4.23 (1H, m, CH), 4.11 (1H, m, CH), 4.07 (4H, broad s, N(CH 2 ) 2 ), 3.51 (1H, t, CH), 3.27 (1H, s, CH), 1.81 (4H, broad d, C(CH 2 ) 2 ), 1.22 (3H, d, CH 3 ) ppm ppm.

Anal. Calcd. for C 17 H 16 N 4 O 5 S: C, 52.57; H, 4.15; N, 14.43; S, 8.26

Found: C, 52.42; H, 4.08; N, 14.50; S, 8.27

›Example 11

2-(1,5-dioxa-9-azaspiro[5.5]undec-9-yl)-8-nitro-4-oxo-1,3-benzothiazine-6-carbonitrile, (compound 11)

Following the procedure of Example 9. Yellow crystalline solid. Yield 40%. mp 230-232° C. (EtOH/DMF).

R f (hexane-acetone; 2/1)-0.15.

MS m/z 388 (M + ).

1 H NMR (DMSO-d 6 ) δ 8.74 and 8.61 (two 1H, two s, 2CH), 3.29-3.65 (6H, m, 3CH 2 ), 2.38 (4H, broad s, 2CH 2 ), 1.82-1.93 (4H, m, 2CH 2 ) ppm.

Anal. Calcd. for C 17 H 16 N 4 O 5 S: C, 52.57; H, 4.15; N, 14.43; S, 8.26

Found: C, 52.52; H, 4.11; N, 14.59; S, 8.13

›Example 12

Determination of the In Vitro Inhibitory Activity of the Compounds of the Invention Against Mycobacteria

The antibacterial activities of the compounds against Mycobacterium smegmatis SG 987 , M. aureum SB66, M. vaccae IMET 1010670 and M. fortuitum B were tested by determination of minimal inhibitory concentrations (MIC) by the micro broth dilution method in Mueller-Hinton broth (Difco) according to the NCCLS guidelines [National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; 5 th Ed.; Villanova, Ed.; Approved standard Document M7-A5. NCCLS, (2000)]

Activity against M. tuberculosis H37Rv was tested by the following method for determination of minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC):

Strains were inoculated onto solid Lowenstein-Jensen medium. After 21 days, the cultures grown were used to prepare an inoculum suspension corresponding to 5×10 8 microbial cells/ml). With 0.2 ml of that suspension tubes with 2 ml liquid Shkolnikova medium, containing corresponding concentrations of compounds under study—from 100.0 to 0.195 Mg/ml, were inoculated. After 14 days of incubation at 37° C. the tubes with liquid medium were centrifuged for 15 min. at 3000 RPM.

After discarding the supernatant, the sediment was resuspended in 0.8 ml of sterile 0.9% NaCl. 0.1 ml of the suspension was used to prepare smears subsequently stained by the Ziehl-Neelsen method. The remaining sediment was inoculated in 0.2 ml volumes into three tubes with solid drug free Lowenstein-Jensen medium to determine minimal bactericidal concentrations (MBC). The results were read after 21-28 days of cultivation at 37° C. Controls were tubes cultured with test-strains not treated with the studied agents.

Minimal bactericidal concentration of drugs (MBC) was considered as the drug concentration completely inhibiting the growth of mycobacteria on the solid medium. The bacteriostatic effect (MIC) was characterized by the presence of only individual mycobacteria in the smear and a strong decrease in the number of colonies grown on solid media compared to the controls.

The results are presented in Tables 1 and 2.

›Example 13

Determination of the In Vivo Inhibitory Activity of the Compounds of the Invention Against Mycobacterium tuberculosis in the Murine TB Model

To determine the chemotherapeutic efficacy we used BALB/c line mice with experimental hematogenously disseminated tuberculosis. The mice were obtained from the Central Animal Nursery of the Russian Academy of Medical Sciences. In this study we included mice after quarantine, standardized by weight (20-25 g) and male only. The mice were infected with a 2-week virulent culture of Mycobacterium tuberculosis H37Rv by intravenous injection (into tail vein) of the mycobacterial suspension at a dose of 5×10 6 CFU (Colony Forming Unit) in 0.5 ml saline. All the experimental animals were divided into groups depending on the treatment regimen used (Table 3). Tested drug doses were selected based on the data from literature and on results of previous investigations.

Treatment was started the next day after infection. The drugs were introduced orally as suspension in carboxymethylcellulose/water with a small quantity PEG-400.

Chemotherapy was administered daily 6 times per week (except Sunday).

The animals were killed with ether narcosis. To determine the efficacy of each treatment regimen we registered macroscopical changes in parenchymal organs of the mice, growth of mycobacteria from pathologic material on solid media, as well as a bacterioscopical index of organ injury. We carried out a qualitative and quantitative analysis of macroscopical changes in the liver, spleen and lungs and calculated an injury index (using a four-score scale).

Macroscopical evaluation of the efficacy of each treatment regimen was expressed in the efficacy index, calculated using a formula.

Microbiological examination included culture for determination of CFU in parenchymal organs. For this purpose, we homogenisated the right lung and separately the spleen with 6% sulfuric acid, centrifuged, washed by water and saline. The yield (about 0.5 mL) was diluted by 1.0 mL of saline and homogenisated. This suspension (0.5 mL) of test organs was diluted 100 and 1000 times by saline and was distributed on solid Finn-2 medium. The cultures were incubated at 37° C. for 1 months and read weekly starting from day 10. After 28 days CFU's were counted.

Data of macroscopical and microbiological examinations of parenchymal organs of mice which died during the experiment were also considered in the overall assessment of the experimental results which are represented in tables 4-6.

All control animals died until day 33

›Tables in the description — 6

TABLE 1 — Antimicrobial activity of compounds as of the formula I determined by minimal inhibitory concentrations MIC [μg/ml] Compound. M. smegmatis M. vaccae M. fortuitum

1	12.5	ng/ml	3.12	ng/ml	12.5	ng/ml
2	1.56	ng/ml	0.76	pg/ml	0.023	pg/ml
3	0.2	μg/ml	0.0015	μg/ml	0.006	μg/ml
4	0.2	μg/ml	0.003	μg/ml	0.003	μg/ml
5	6.25	ng/ml	0.078	ng/ml	0.078	ng/ml
6	>10	μg/ml	0.04	μg/ml	0.08	μg/ml
7	0.78	μg/ml	0.003	μg/ml	0.003	μg/ml
8	0.4	μg/ml	0.025	μg/ml	0.025	μg/ml
9	0.05	μg/ml	3.12	ng/ml	25	ng/ml
10	25	ng/ml	3.12	ng/ml	12.5	ng/ml
11	0.05	μg/ml	6.25	ng/ml	25	ng/ml

TABLE 3

No		Dose	Number of animals
group	Compound	(mg/kg)	per group
3	2	12	10
4	2	25	10
5	Isoniazid (INH)	25	10
6	without treatment		10

Efficacy	⁢
⁢	index
=
100	⁢	%
-
Injury	⁢
⁢	index	⁢
⁢	of	⁢
⁢	the
studied	⁢
⁢	group
Injury	⁢
⁢	index	⁢
⁢	of	⁢
⁢	the
control	⁢
⁢	group
×	100

TABLE 4 — Indexes of organ injury in mice and treatment efficacy Dose

Group	Drug	(mg/kg)	Injury index	Efficacy index (%)
3	Compound 2	12	2.1	44.7
4	Compound 2	25	1.0	78
5	INH, Isoniazid	25	1.2	70.5
6	Control	—	3.8	—

TABLE 5 — Results of microbiological examination of right lung and spleen of experimental mice (42 days after inoculation of the culture medium)

			right lung	spleen
			Culture, without	Culture, without
		Dose	dilution	dilution
Group	Compound	(mg/kg)	CFU	CFU
3	2	12	~60	~60
4	2	25	~35	~35
5	INH, Isoniazid	25	~40	~40
6	Control	—	>120	>120
			(total growth)	(total growth)

TABLE 6 — Survival of animals

Day of	Group 3	Group 4	Group 5	Group 6
Treatment	Compound 2	Compound 2	INH	Control
1	10	10	10	10
2	10	10	10	10
3	10	10	10	10
4	10	10	10	10
5	10	10	10	10
6	10	10	10	10
7	10	10	10	10
8	10	10	10	10
9	10	10	10	10
10	10	10	10	10
11	10	10	10	10
12	10	10	10	10
13	10	10	10	9
14	10	10	10	9
15	10	10	10	9
16	10	10	10	9
17	10	10	10	9
18	10	10	10	9
19	10	10	10	9
20	10	10	10	8
21	10	10	10	8
22	10	10	10	8
23	10	10	10	8
24	10	10	10	8
25	10	10	10	5
26	10	10	10	4
27	100%	100%	100%	40%

Claims

11 · 5 independent · depth 2

11 granted claims

›1.independentA compound of formula (I)+6

›5.independentA compound selected from the group consisting of:n2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one,2-(7,12-dio…

›6.independent2-(2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-8-nitro-6-(trifluoromethyl)-1,3-benzothiazin-4-one.

›9.independentA method of treating tuberculosis in a mammal, comprising administering to the mammal an effective amount of a pharmaceutical composition comprising a…

›10.independentmultiple · 2, 3, 4, 5A method for the treatment of a disease selected from tuberculosis and leprosy in a mammal comprising the step of administering to a mammal in need of…

Classifications

5 codes

IPC · International Patent Classification

Section A — Human necessities

A61K31/541

Section C — Chemistry; metallurgy

C07D417/06

USPC · US Patent Classification

514/224.2544/6544/52

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Pendency

4.6 y

1,686 days filing → grant

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Kahsay T Habte

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Priority chain

1 priority documents

›Priority documents — 1

Type	Document	Date
related publication	US 20090239851 A1	24 Sep 2009

Worldwide family

37 members · 24 offices

this patentIP5 & PCTother officessolid = grantedhover for detail · click to open

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DOCDB simple family 37595033

Offices

US · EP · JP · KR · CN · WO

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11 of 37

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Non-English titles

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›IP5 & PCT — 12 members

Office	Publication	Kind	Published	Filed	Status	Title
US	US-2009239851-A1	A1	24 Sep 2009	24 May 2006	published	Benzothiazinone Derivatives and their Use as Antibacterial Agents
USthis patent	US-7863268-B2	B2	4 Jan 2011	24 May 2006	granted	Benzothiazinone derivatives and their use as antibacterial agents
US	US-2011160193-A1	A1	30 Jun 2011	22 Dec 2010	published	New Benzothiazinone Derivatives and Their Use as Antibacterial Agents
EP	EP-2029583-A1	A1	4 Mar 2009	24 May 2006	published	Nouveaux dérivés de la benzothiazinone et leur utilisation en tant qu'agents antibactériensfr
EP	EP-2029583-B1	B1	9 Jun 2010	24 May 2006	granted	Neue benzothiazinonderivate und ihre verwendung als antibakterielle mittelde
JP	JP-2009537576-A	A	29 Oct 2009	24 May 2006	published	新規なベンゾチアジノン誘導体、及び抗菌剤としてのその使用ja
JP	JP-5068813-B2	B2	7 Nov 2012	24 May 2006	granted	新規なベンゾチアジノン誘導体、及び抗菌剤としてのその使用ja
KR	KR-20090029736-A	A	23 Mar 2009	24 May 2006	published	신규한 벤조티아지논 유도체 및 그의 항균제로서의 용도ko
KR	KR-101314999-B1	B1	4 Oct 2013	24 May 2006	granted	New benzothiazinone derivatives and their use as antibacterial agents
CN	CN-101472922-A	A	1 Jul 2009	24 May 2006	published	Novel benzo buprofezin derivate and application thereof as antibacterial agent
CN	CN-101472922-B	B	30 May 2012	24 May 2006	granted	苯并噻嗪酮衍生物及其作为抗菌剂的应用zh
WO	WO-2007134625-A1	A1	29 Nov 2007	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents

›Other offices — 25 members

Office	Publication	Kind	Published	Filed	Status	Title
AP	AP-2008004723-A0	A0	31 Dec 2008	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents
AP	AP-2444-A	A	31 Aug 2012	24 May 2006	granted	New benzothiazinone derivatives and their use as antibacterial agents
AT	AT-E470667-T1	T1	15 Jun 2010	24 May 2006	granted	Neue benzothiazinonderivate und ihre verwendung als antibakterielle mittelde
AU	AU-2006343832-A1	A1	29 Nov 2007	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents
AU	AU-2006343832-B2	B2	21 Jul 2011	24 May 2006	granted	New benzothiazinone derivatives and their use as antibacterial agents
BR	BR-PI0621693-A2	A2	20 Dec 2011	24 May 2006	published	compostos derivados de benzotiazinona, composição farmacêutica, bem como seu uso como agentes antibacterianospt
BR	BR-PI0621693-B1	B1	17 Nov 2020	24 May 2006	published	compostos derivados de benzotiazinona, composição farmacêutica, e seu uso como agentes antibacterianospt
BR	BR-PI0621693-B8	B8	25 May 2021	24 May 2006	published	compostos derivados de benzotiazinona, composição farmacêutica, e seu uso como agentes antibacterianospt
CA	CA-2652841-A1	A1	29 Nov 2007	24 May 2006	published	Nouveaux derives de la benzothiazinone et leur utilisation en tant qu'agents antibacteriensfr
CA	CA-2652841-C	C	27 Jan 2015	24 May 2006	granted	New benzothiazinone derivatives and their use as antibacterial agents
CR	CR-10519-A	A	14 Apr 2009	17 Dec 2008	published	Nuevos derivados de benzotiazinona y su uso como agentes antibacterianoses
CY	CY-1110769-T1	T1	10 Jun 2015	7 Sep 2010	published	Νεα παραγωγα βενζοθειαζιnονης και η χρηση τους ως αντιβακτηριακων παραγοντωνel
DE	DE-602006014885-D1	D1	22 Jul 2010	24 May 2006	published	Neue benzothiazinonderivate und ihre verwendung als antibakterielle mittelde
DK	DK-2029583-T3	T3	27 Sep 2010	24 May 2006	granted	Nye benzothiazinonderivater og anvendelse heraf som antibakterielle midlerda
EA	EA-200870564-A1	A1	30 Jun 2009	24 May 2006	published	Новые производные бензотиазинона и их применение в качестве антибактериальных средствru
EA	EA-015910-B1	B1	30 Dec 2011	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents
ES	ES-2345902-T3	T3	5 Oct 2010	24 May 2006	granted	Nuevos derivados de benzotiazinona y su uso como agentes antibacterianos.es
IL	IL-195453-A0	A0	3 Aug 2009	23 Nov 2008	published	New benzothiazinone derivatives and their use as antibacterial agents
IL	IL-195453-A	A	31 Jan 2013	23 Nov 2008	published	Benzothiazinone derivatives, process for their preparation and pharmaceutical compositions comprising them
MX	MX-2008014958-A	A	15 Apr 2009	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents.
NZ	NZ-573833-A	A	29 Oct 2010	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents
PL	PL-2029583-T3	T3	29 Oct 2010	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents
PT	PT-2029583-E	E	12 Aug 2010	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents
SI	SI-2029583-T1	T1	30 Sep 2010	24 May 2006	published	New benzothiazinone derivatives and their use as antibacterial agents
TN	TN-SN08474-A1	A1	14 Apr 2010	21 Nov 2008	published	New benzothiazinone derivatives and their use as antibacterial agents

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